Journal: bioRxiv

Article Title: A chemically defined and xeno-free hydrogel system for regenerative medicine

doi: 10.1101/2024.05.28.596179

Figure Lengend Snippet: (A) ELISA of culture medium for Apolipoprotein B (APOB), taken from iHeps 2 days after the end of the hepatic differentiation protocol, and from PHH culture media 2 days after plating. (B) Brightfield microscopy of 3D tubular branching strictures observed when Laminin 411 concentrations are increased. Scale bar = 100μm. (C) Alkaline Phsophatase (ALP) activity measured by colourimetric assay on culture medium taken 2 days the end of the hepatic differentiation protocol.

Article Snippet: APOB secretion in the supernatant was quantified using the human APOB ELISA quantification kit (Mabtech, # 3715-1H-6) according to product literature.

Techniques: Enzyme-linked Immunosorbent Assay, Microscopy, Activity Assay

Journal: Bone

Article Title: ACVR1 R206H extends inflammatory responses in human induced pluripotent stem cell-derived macrophages.

doi: 10.1016/j.bone.2021.116129

Figure Lengend Snippet: Fig. 1. Differentiation of 3D-iMACs and their functional analysis. A. Schematic representation of the protocol for generating 3D-iMACs. All cell culture was performed under hypoxic (5% O2) conditions. B. Bright-filed images at day 4, 10, 17, and 24. Scale bar, 200 μm. C. Surface marker expressions of 3D-iMACs at day 24. Most cells express both of CD163 and CD206. D. Comparison of cytokine production profiles between 3D-iMACs and primary M2-like macrophages. They were not treated (NT) or stimulated with 10 ng/ml LPS for 24 h (LPS). n = 3 biological replicates. iMACs and primary MACs showed similar cytokine profiles with or without LPS stimulation. E. Phagocytic activities of 3D-iMACs were confirmed under a fluorescent microscope. E. coli BioParticles conjugated with pHrodo Green appear bright green when they are taken up by macrophages. Scale bar, 200 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: On day 4, medium was removed in the same way as described above and cultured on a new low-attachment plate in medium consisting of PromoCell basal media (PromoCell) supplemented with Cytokine Mix E (PromoCell), 10 ng/ml VEGF (Calbiochem), 10 ng/ml bFGF (Peprotech), 25 ng/ml IL-6 (Peprotech), and 10 ng/ml IL-11 (Peprotech).

Techniques: Functional Assay, Cell Culture, Marker, Comparison, Microscopy

Journal: Bone

Article Title: ACVR1 R206H extends inflammatory responses in human induced pluripotent stem cell-derived macrophages.

doi: 10.1016/j.bone.2021.116129

Figure Lengend Snippet: Fig. 2. Differentiation of 2D-iMACs and their functional analysis. A. Schematic representation of the protocol for generating 2D-iMACs. B. Bright-field images at days 12 and 19. Scale bar, 200 μm. C. Surface marker expression of 2D- iMACs at days 12 and 19 (WT1323). They are positive for CD45, CD14, and CD11b. D. Polarization protocol and their subsequent surface marker expression. While M2-like iMACs showed high CD163 and low CD80 positivity, the expression patterns were opposite in M1-like iMACs. E. mRNA expression levels of macrophage- related genes in M1-like and M2-like 2D-iMACs. M1-related and M2-related genes are shown in the upper and lower portions respectively. They showed the expected expression patterns except for TGF-β. Expression levels are normalized to the housekeeping gene β-actin. Student's t-test was used for comparison of two groups. **p < 0.01. Data represent mean ± SEM of four independent experiments with technical triplicates. F. Phagocytic activities of M1-like and M2-like 2D-iMACs were confirmed (WTC11). E. coli BioParticles taken up by macrophages appear bright green. Scale bar, 200 μm. G. Comparison of cytokine production profiles between 2D- iMACs and primary macrophages. M1-like and M2-like 2D-iMACs showed similarities to primary M1- and M2-like macrophages respectively. n = 4–6 biological replicates. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: On day 4, medium was removed in the same way as described above and cultured on a new low-attachment plate in medium consisting of PromoCell basal media (PromoCell) supplemented with Cytokine Mix E (PromoCell), 10 ng/ml VEGF (Calbiochem), 10 ng/ml bFGF (Peprotech), 25 ng/ml IL-6 (Peprotech), and 10 ng/ml IL-11 (Peprotech).

Techniques: Functional Assay, Marker, Expressing, Comparison

Journal: Bone

Article Title: ACVR1 R206H extends inflammatory responses in human induced pluripotent stem cell-derived macrophages.

doi: 10.1016/j.bone.2021.116129

Figure Lengend Snippet: Fig. 4. Response to PAMP (LPS) and DAMPs (HMGB1, S100A8/A9) stimulation in 2D-iMACs. A. Protocol for testing the response to PAMPs and DMAPs. B. Comparison of cytokine production profiles in response to a PAMP (LPS) and DAMPs (HMGB1, S100A8/ A9) between primary M1-like macrophages and M1-like 2D iMACs. Primary cells and iMACs showed similarities regarding their responses to LPS. NT, HMGB, and S100 labels refer to untreated, stimulated with HMGB1, and stimulated with S100A8/A9, respectively. Primary cells and iMACs showed strong similarities regarding their responses to LPS. C. Comparison of cytokine production profiles in response to PAMP (LPS) and DAMPs (HMGB1, S100A8/A9) between primary M2-like macrophages and M2-like 2D iMACs. n = 2–3 biological replicates for each condition.

Article Snippet: On day 4, medium was removed in the same way as described above and cultured on a new low-attachment plate in medium consisting of PromoCell basal media (PromoCell) supplemented with Cytokine Mix E (PromoCell), 10 ng/ml VEGF (Calbiochem), 10 ng/ml bFGF (Peprotech), 25 ng/ml IL-6 (Peprotech), and 10 ng/ml IL-11 (Peprotech).

Techniques: Comparison

Journal: Bone

Article Title: ACVR1 R206H extends inflammatory responses in human induced pluripotent stem cell-derived macrophages.

doi: 10.1016/j.bone.2021.116129

Figure Lengend Snippet: Fig. 5. Comparison between WT-iMACs and FOP-iMACs. A. Comparison of cytokine production profiles between WT and FOP 2D-iMACs. Cells were not treated (NT) or stimulated with 10 ng/ml LPS for 24 h (LPS) based on the protocol shown in Fig. 4A. n = 6 biological replicates. Differences between WT- and FOP-iMACs were more apparent in M1-like iMACs. B. Cytokines that showed higher production in FOP-M1-like iMACs compared with WT-M1-like iMACs at their baseline state (nontreated, NT). Student's t-test was used for comparison of two groups. *p < 0.05, **p < 0.01. Data represent mean ± SEM of six independent experiments with technical triplicates. C. Cytokine concentrations showing significant differences between WT- and FOP-M1-like iMACs stimulated with 10 ng/ml LPS. Key pro-inflammatory cytokine concentrations were higher in WT-M1-like iMACs when stimulated with 10 ng/ml LPS. Student's t-test was used for comparison of two groups. **p < 0.01Data represent mean ± SEM of six independent experiments with technical triplicates.

Article Snippet: On day 4, medium was removed in the same way as described above and cultured on a new low-attachment plate in medium consisting of PromoCell basal media (PromoCell) supplemented with Cytokine Mix E (PromoCell), 10 ng/ml VEGF (Calbiochem), 10 ng/ml bFGF (Peprotech), 25 ng/ml IL-6 (Peprotech), and 10 ng/ml IL-11 (Peprotech).

Techniques: Comparison

Journal: Bone

Article Title: ACVR1 R206H extends inflammatory responses in human induced pluripotent stem cell-derived macrophages.

doi: 10.1016/j.bone.2021.116129

Figure Lengend Snippet: Fig. 6. Activin A concentrations in iMACs and cytokine concentrations in FOP-M1-like iMACs treated with inhibitors of Activin A pathways. A. Activin A concentrations in NT and LPS-stimulated group were analyzed using Human/Mouse/Rat Activin A Quantikine ELISA Kit. Analyzed samples were the same as those used in Fig. 5A. The concentrations of FOP-M1-like iMACs were significantly higher than WT-M1-like iMACs in NT group, while their response to LPS was downregulated. Student's t-test was used for comparison of two groups. *p < 0.05, **p < 0.01. Data represent mean ± SEM of six independent experiments with technical triplicates. B. FOP-M1-like iMACs were treated with 100 ng/ml anti-human/mouse/rat Activin A antibody (Anti) or 10 mM SB431542 (SB) after M1 polarization. Each inhibitor was added every 24 h. After 3 days culture, supernatants were collected and analyzed. There were no significant differences in key pro- inflammatory cytokines that were found elevated in FOP-M1-like iMACs as shown in Fig. 5B. Dunnett's test was used to compare each group to control (CTRL). Data represent mean ± SEM of five independent experiments with technical triplicates.

Article Snippet: On day 4, medium was removed in the same way as described above and cultured on a new low-attachment plate in medium consisting of PromoCell basal media (PromoCell) supplemented with Cytokine Mix E (PromoCell), 10 ng/ml VEGF (Calbiochem), 10 ng/ml bFGF (Peprotech), 25 ng/ml IL-6 (Peprotech), and 10 ng/ml IL-11 (Peprotech).

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: ACS infectious diseases

Article Title: Tyrosine-Based Cross-Linking of Peptide Antigens to Generate Nanoclusters with Enhanced Immunogenicity: Demonstration Using the Conserved M2e Peptide of Influenza A.

doi: 10.1021/acsinfecdis.1c00219

Figure Lengend Snippet: Figure 6. In vitro antigenicity analysis of t-M2e-t NCs using ELISA. (A) Absorbance values from an indirect ELISA performed by coating 96-well plates with a fixed amount of either un-cross-linked t-M2e-t peptide, cross-linked t-M2e-t NCs, or a negative control peptide (NCP). Mouse serum collected from mice vaccinated with a gold nanoparticle and M2e formulation (from another study8−10) was used as a source of M2e detection IgG antibody, which was applied at different dilutions. (B) Absorbance values from a sandwich ELISA performed by coating 96-well plates with a fixed amount of purified mouse anti-M2e IgM monoclonal antibody as capture antibody. Dilutions of analyte (either un-cross-linked t-M2e-t peptide, cross-linked t-M2e-t NCs, or a NCP) were then added to the wells. Mouse serum collected from mice vaccinated with a gold nanoparticle and M2e formulation (from another study8−10) was used as a source of primary M2e detection IgG antibody. Cartoons of each ELISA procedure are shown below their respective graphs.

Article Snippet: All peptides were end-terminal acetylated and amidated in order to increase peptide stability against degradation.65−67 Goat anti-mouse IgG (1030−05), goat anti-mouse IgG1 (1070−05), and goat anti-mouse IgG2a (1080−05) secondary antibodies with HRP were obtained from Southern Biotech (AL, USA).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Indirect ELISA, Negative Control, Formulation, Sandwich ELISA

Journal: ACS infectious diseases

Article Title: Tyrosine-Based Cross-Linking of Peptide Antigens to Generate Nanoclusters with Enhanced Immunogenicity: Demonstration Using the Conserved M2e Peptide of Influenza A.

doi: 10.1021/acsinfecdis.1c00219

Figure Lengend Snippet: Figure 7. Immune response and survival data of mice after immunization with t-M2e-t NCs. Mice were immunized thrice, once each on day 0, 21, and 42. Blood was collected and analyzed for anti-M2e antibodies. Within 1 week of the final serum collection, mice were challenged with 3× LD50 A/California/07/2009 (H1N1) and monitored for 14 days. Two antigens were used (i) 20 μg or 5 μg t-M2e-t UCP (un-cross-linked peptide) with/without 20 μg CpG, and (ii) 20 μg or 5 μg S t-M2e-t NCs (small NCs) with/without 20 μg CpG. (A−C) Anti-M2e IgG, IgG1 and IgG2a titers with t-M2e-t UCP as antigen. (D−F) Anti-M2e IgG, IgG1 and IgG2a titers with t-M2e-t NC as antigen. (G, H) Body weight and survival after virus challenge of t-M2e-t UCP groups. (I, J) Body weight and survival after virus challenge of t-M2e-t NP groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: All peptides were end-terminal acetylated and amidated in order to increase peptide stability against degradation.65−67 Goat anti-mouse IgG (1030−05), goat anti-mouse IgG1 (1070−05), and goat anti-mouse IgG2a (1080−05) secondary antibodies with HRP were obtained from Southern Biotech (AL, USA).

Techniques: Virus